1. Field of the Invention
The present invention relates generally to methods and compositions for replacement gene therapy, and more particularly relates to adenovirus vectors adapted for delivering functional apolipoprotein A-I (apoA-I) genes to liver cells. It is proposed that the methods and compositions disclosed herein will be applicable to elevating the HDL (high density lipoprotein) form of cholesterol and thus suitable for use in treating atherosclerosis and reducing cardiovascular risk.
2. Description of the Related Art
Epidemiologic data demonstrate an inverse relationship between circulating levels of high density lipoprotein cholesterol (HDL cholesterol) and the incidence of clinically significant atherosclerosis (Miller, 1987; Manninen et al., 1988; Kottke et al., 1986; Gordon et al., 1989). This relationship holds for even small increments of HDL cholesterol, such that each 1 mg/dl increase in HDL cholesterol level is associated with a 2-3% decrement in cardiovascular risk (Gordon et al., 1989). Experimental evidence also supports a protective effect of HDL against atherosclerosis. Cholesterol-fed rabbits treated by infusion of purified homologous HDL are protected against the development of fatty plaques despite unchanged circulating HDL cholesterol levels (Badimon et al., 1989; Badimon et al., 1990; Badimon et al., 1992). This association between HDL cholesterol and the incidence of atherosclerotic vascular disease suggests that strategies to increase circulating HDL could have important clinical application. A modest increase in HDL cholesterol has been observed in patients treated with gemfibrozil (Badimon et al., 1989), an intervention associated with a reduced incidence of cardiac events. Trials intended to specifically assess the effects of intervention to increase HDL cholesterol on the development and progression of atherosclerosis are in progress (Goldbourt et al., 1993; Rubins et al., 1993).
HDL appears to exert its antiatherogenic effect by mediating reverse cholesterol transport, in which cholesterol is mobilized from peripheral tissues and transported to the liver (Eisenberg, 1984; Reichl et al., 1986; Miller, 1990). The small, high density, pre-beta subspecies of HDL, comprised predominantly of apolipoprotein A-1 and phospholipid is thought to act as the physiologic acceptor for cholesterol in the extracellular matrix of peripheral tissues (Reichl et al., 1986). Peripheral availability of this xe2x80x9cscavengerxe2x80x9d particle appears to be regulated by the rates of synthesis, secretion and catabolism of HDL (Eisenberg, 1984; Reichl et al., 1986; Miller, 1990).
Both clinical and experimental data suggest that the principal protein constituent of HDL, apolipoprotein A-1, mediates the antiatherogenic activity of HDL (Miller, 1987), and that the rate of production of apoA-I is a critical determinant of circulating HDL cholesterol. Families with both heritably deficient (Karathanasis et al., 1983; Vergani et al., 1981; Third et al., 1984; Ordovas et al., 1986) and enhanced (Glueck et al., 1976) apolipoprotein A-1 levels have been identified, and show corresponding alterations in HDL cholesterol. Persons with familial hyperalphalipoproteinemia appear protected from atherosclerosis, while those deficient in apolipoprotein A-1 show accelerated cardiovascular disease. Mice transgenic for a copy of the human apolipoprotein A-1 gene demonstrate accumulation of human apoA-1 in serum, increased circulating HDL cholesterol, and resistance to the atherogenic effects of a high cholesterol diet (Rubin et al., 1991; Walsh et al., 1989; Sorci-Thomas et al., 1988; Rubin et al., 1991). Thus, while the mechanisms regulating the rate of apolipoprotein A-1 synthesis are not clearly defined, genetic factors appear to exert an important effect (Widom et al., 1991).
A potential approach to increasing levels of apolipoprotein A-1 is somatic cell gene therapy. Recently, adenovirus-mediated gene transfer has been investigated as a means of mediating gene transfer into eukaryotic cells and into whole animals (van Doren et al., 1984a; van Doren et al., 1984b; Ghosh-Choudhury and Graham, 1987; Stratford-Perricaudet et at., 1990; Rosenfeld et al., 1991; Rosenfeld et al., 1992). Stratford-Perricaudet et al. (1990) have shown that adenovirus-mediated gene transfer can be used to treat a rare recessive genetic disorder, ornithine transcarbamylase (OTC) deficiency, in newborn mice. Unfortunately, the expression of the ornithine transcarbamylase enzyme in the virus injected mice was comparable to that in normal mice in only 4 out of 17 instances. In one out of 17 instances the level was about half the normal level, and in the remaining 12 out of 17, it was less than 20% of normal. Therefore, the defect was only partially corrected in most of the mice and led to no phenotypic or physiologic change in those mice.
Attempts to use adenovirus to transfer the gene for cystic fibrosis transmembrane conductance regulator (CFTR) into the pulmonary epithelium of cotton rats have also been successful, although it has not been possible to assess the biological activity of the transferred gene in the epithelium of the animals (Rosenfeld et al., 1992). Again, these studies demonstrated gene transfer and expression of the CFTR protein in lung airway cells but showed no physiologic effect. In the 1991 Science article, Rosenfeld et al. showed lung expression of xcex11-antitrypsin protein but again showed no physiologic effect. In fact, they estimated that the levels of expression that they observed were only about 2% of the level required for protection of the lung in humans, i.e., far below that necessary for a physiologic effect. These results therefore do not demonstrate that adenovirus is able to transfer genes into cells and direct the expression of sufficient protein to achieve a physiologically relevant effect, and would not suggest a usefulness of the adenovirus system for use in connection with apo A-1 gene therapy.
Similarly, the gene for human xcex11-antitrypsin has been introduced into the liver of normal rats by intraportal injection, where it was expressed and resulted in the secretion of the introduced human protein into the plasma of these rats (Jaffe et al., 1992). However, the levels that were obtained were not high enough to be of therapeutic value.
In an alternate approach, a plasmid construct which encodes the human ApoA1 gene has been encapsulated in liposomes and introduced into the liver of rats by direct injection (Frolkis et al., 1991). This method resulted in increased HDL levels in the animals. However, the procedure is invasive, requiring anesthesia and an incision in the abdominal wall in order to introduce the liposome suspension directly into the liver.
Thus, there is clearly a significant need for novel therapeutic approaches that would be applicable to the treatment of diseases involving atherosclerosis. There is a particular need for the development of approaches that can lead to significant increases in HDLc. There is also a particular need for treatment methodologies that do not require surgical intervention, such as direct injection into the liver or modification of hepatocytes ex vivo.
The present invention addresses one or more of these or other shortcomings in the prior art through the provision of an adenovirus mediated technique for introducing human apoA-1 coding sequences into eukaryotic cells and expression and secretion in liver cells without the need for surgical intervention. The technique of the present invention circumvents many of the problems of the currently available techniques, and is based upon the discovery by the inventors that adenovirus vectors can selectively deliver apoA-1 coding sequences to liver cells and effect expression therein, and thereby achieve a physiologically significant effect.
In view of these observations, somatic cell gene transfer to augment apolipoprotein A-1 expression offers a new and potentially effective therapeutic approach. In an embodiment of the present invention, normal mice infected with a recombinant adenovirus encoding human apolipoprotein A-1 express high levels of human apoA-1 in serum. These animals demonstrate increases in circulating HDL cholesterol similar to those observed in mice transgenic for a copy of the human apolipoprotein A-1 gene, and of a magnitude previously associated with a protective effect against the development and/or progression of experimental atherosclerosis.
The invention generally relates to an adenovirus vector construct which includes a human apoA-1 expression region recombinant insert that is capable of expressing human apoA-1 in transformed cells. As used herein, a human apoA-1 expression region recombinant insert is defined as a DNA sequence that encodes the mature human apoA-1 protein, as, for example disclosed in Karathanasis et al., 1983 and Law and Brewer, 1984 joined to, 3xe2x80x2 of and in frame with, the secretory signal sequence from human tissue plasminogen activator (tPA), for example. The expression region may also comprise a promoter and a polyadenylation site. In its most preferred embodiment, the vector is vector AdCMVapoA-1 as constructed by the methods disclosed hereinbelow in Example 6. While for ease of use one will prefer to employ a sequence derived from an apoA-1 cDNA sequence, it is contemplated that genomic sequences may be employed where desired.
The practice of the present invention rests in part upon the discovery that adenovirus vectors have been found by the inventors to selectively direct recombinant expression coding sequences to liver cells, and that these are efficiently expressed in the liver. The adenovirus vectors of the present invention have been rendered replication defective through deletion of the viral early region 1 (E1A) region such that the virus is competent to replicate only in cells, such as human 293 cells, which express adenovirus early region 1 genes from their cellular genome. This is important because the virus will therefore not kill normal cells because these cells do not express early gene products. Techniques for preparing replication defective adenoviruses are well known in the art as exemplified by Berkner et al., 1983, Ghosh-Choudhury et al., McGrory et al., 1988, and Gluzman et al.; see also U.S. Ser. No. 07/823,747, filed Jan. 22, 1992, incorporated herein by reference).
The examples of preferred embodiments disclosed herein utilize human adenovirus type 5. Type 5 virus was selected because a great deal of biochemical and genetic information about the virus is known, and it has historically been used for most constructions employing adenovirus as a vector. It is understood, however, the adenovirus may be of any of the 42 different known serotypes of subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the method of the present invention.
Any of a large number of promoters may be used to direct expression the apoA-1 gene. In the examples given, the human cytomegalovirus (CMV) immediate early gene promoter has been used (Thomsen et al., 1984), which results in the constitutive, high-level expression of the foreign gene. However, the use of other viral or mammalian cellular promoters which are well-known in the art is also suitable to achieve expression of the apoA-1, provided that the levels of expression are sufficient to achieve a physiologic effect.
By employing a promoter with well-known properties, the level and pattern of expression of apoA-1 following infection can be optimized. For example, selection of a promoter which is active specifically in liver cells (such as the xcex11-antitrypsin, apolipoprotein A-1, liver fatty acid binding protein, LDL receptor, or plasminogen activator inhibitor type 1 (PAI-1) gene promoters) will permit tissue-specific expression of the apoA-1. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the apoA-1. For example, with a recombinant adenovirus in which the reporter coding sequence of xcex2-galactosidase is expressed from the human PAI-1 promoter, Applicants have found that xcex2-galactosidase expression can be induced in endothelial cells by tumor necrosis factor.
The secretory signal sequence to be used in the practice of the present invention may be any signal sequence that will direct the proper secretion of the apoA-1 protein. The human tissue plasminogen activator (tPA) secretory signal sequence was chosen because of the convenience of the location of restriction enzyme recognition sites that were compatible with the one in the propeptide cleavage site from human apolipoprotein A-1. It is understood that any signal sequence which can be joined to the apoA-1 gene in frame and which will direct the secretion and maturation of a propeptide from a mammalian cell in an efficient manner is acceptable; however, certain secretory signal sequences may have advantages under different conditions and one will select these depending on the particular circumstances. Alternative secretory signal sequences which may be used include, but are not limited to human PA1-1 or the endogenous apoA-1 sequences with the human tissue plasminogen activator signal sequence being the most preferred.
The vectors of the present invention are replication defective, and as such they will typically not have an adenovirus E1 region. Thus, it will be most convenient to introduce the apoA-1 coding region at the position from which the E1 coding sequences have been removed. However, the apoA-1 coding region may be inserted in other regions as long as it is expressed. The apoA-1 transcription unit may also be inserted, e.g., in the position of the deleted E3 region in E3 replacement vectors as described previously by Karlsson et at. (1986). Moreover, where a cDNA insert is employed one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the apoA-1 message. Any such sequence may be employed. The inventors prefer to employ either the SV40 or protamine gene polyadenylation signal in that they are convenient and known to function well in the target cells employed.
In further embodiments, the invention relates to pharmaceutical compositions wherein the adenovirus vector/apoA-1 gene construct is dispersed in a pharmacologically acceptable solution or buffer. Preferred solutions include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Of course, one will desire to purify the vector sufficiently to render it essentially free of undesirable contaminant, such as defective interfering adenovirus particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the individual receiving the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
In still further embodiments, the invention relates to a method for increasing the plasma high density lipoprotein cholesterol in a subject comprising administering to the subject an effective amount of a pharmaceutical composition which includes the adenovirus vector/apoA-1 construct. Extrapolating from the data set forth hereinbelow, the inventors propose that an effective amount of the vector construct will involve the administration of from about 5xc3x971010 to 5xc3x971012 virus particles, which may be given either as a single bolus injection or as an intravenous infusion over several hours.
In that adenovirus is a virus that infects humans, there may be certain individuals that have developed antibodies to certain adenovirus proteins. In these circumstances, it is possible that such individuals might develop an immunological reaction to the virus. Thus, where an immunological reaction is believed to be a possibility, one may desire to first test the subject to determine the existence of antibodies. Such a test could be performed under a variety of accepted protocols, for example, through a simple skin test or through a test of the circulating blood levels of adenovirus-neutralizing antibodies. In fact, under such circumstances, one may desire to introduce a test dose of on the order of 1xc3x97105 to 1xc3x97106 or so virus particles. Then, if no untoward reaction is seen, the dose may be elevated over a period of time until the desired dosage is reached, such as through the administration of incremental dosages of approximately an order of magnitude.
It should also be pointed out that because the adenovirus vector employed is replication defective, it will not be capable of replicating in the cells that are ultimately infected. Moreover, it has been found that the genomic integration frequency of adenovirus is usually fairly low, typically on the order of about 1%. Thus, where continued treatment in certain individuals is required it may be necessary to reintroduce the virus every 6 months to a year. In circumstances where it may be necessary to conduct long term therapy, the individual""s plasma cholesterol levels are monitored at selected intervals.
The particular cell line used to propagate the recombinant adenoviruses can be any cell which will support replication of the replication deficient virus, or any cell that can supply the E1 region function in trans. The recombinant adenovirus vectors can be propagated on, e.g., human 293 cells, or in other cell lines that are permissive for conditional replication-defective adenovirus infection, e.g., those which express adenovirus E1A gene products xe2x80x9cin transxe2x80x9d so as to complement the defect in a conditional replication-defective vector. Further, the cells can be propagated either on plastic dishes or in suspension culture, in order to obtain virus stocks thereof.